Replication-deficient recombinant adenoviruses are useful, for instance, in gene therapy and for vaccination purposes. They usually lack the E1-region of an adenovirus and are, therefore, propagated on complementing cells providing the E1 sequences. The packaging cells provide all information required for replication of a vector that is packaged in the cells to form recombinant viral particles. An example of a packaging cell is a 293 cell, which contains nucleotides 1-4344 of the adenovirus 5 genome (Louis et al., 1997), which include the 5′ inverted terminal repeat (ITR), the packaging signal, the E1A and E1B coding sequences and the pIX coding sequences.
Overlap between the adenoviral sequences in the packaging cell and the vector may lead to homologous recombination between these sequences, resulting in the generation of replication-competent adenovirus (RCA) (Lochmuller et al., 1994). This problem has been solved by using a packaging system consisting of a cell line and a vector that are matched to each other by lacking such overlapping sequences (Fallaux et al., 1998; U.S. Pat. No. 5,994,128). One example of a particularly useful cell line in such applications is the PER.C6® cell line (U.S. Pat. No. 5,994,128; Nichols et al., 2002).
Recently, it was reported that upon use of PER.C6® cells in conjunction with a vector that still contained 177 bp sequence homology with the E1 sequences in the genome of PER.C6®, a single cross-over event could result in the generation of helper-dependent replication-competent virus at a low frequency (Murakami et al., 2002). The generated atypical RCA was termed “helper-dependent E1-containing particle” (HDEP). As expected, the emergence of this type of particle was circumvented when a vector was used that lacked the sequence overlap with the E1 sequences in the genome of PER.C6® cells (Murakami et al., 2002).
However, it now unexpectedly appears that using the system of matched vector and PER.C6® cells, some batches of recombinant adenovirus are contaminated at a very low frequency with particles that can cause cytopathological effect (CPE) in cells lacking E1 sequences. The particle is helper-dependent and contains E1-sequences and, therefore, is also an HDEP. This HDEP is generated in the absence of substantial homology between vector and packaging cell (see Murakami et al., 2004).
While RCA is recognized by the regulatory authorities as a potential problem and detection of RCA in batches for clinical use is mandatory (USDHHS, FDA, CBER. Guidance for Industry, Guidance for human somatic cell therapy and gene therapy, March 1998), the safety aspects of HDEP are unclear. HDEP is replication deficient since it lacks the necessary viral genes for autonomous replication and, therefore, HDEP will not disseminate in a host. The theoretical possibility that the presence of a recombinant vector or wild-type adenovirus in the same cell may cause rescue and spread of HDEP cannot, however, be completely excluded. Hence, at least a latent need exists for means and methods for preventing generation of E1-containing particles during the preparation of batches of recombinant adenovirus particles. To this purpose, the present invention provides new cell lines, methods for preparing new cell lines, and the use thereof to make batches of RCA-free and HDEP-free recombinant adenovirus vectors.